The Effect Of Metformin Combined With Paclitaxel On Breast Cancer Cells Through Hippo Signaling Pathway

Breast cancer is the most common malignant tumor in women,which is a serious threat to women’s physical and mental health.Although the quality of many cancerpatients lifes having improved with the improvement of diagnosis and treatment technology,chemotherapy resistance of breast cancer still severely limites the control of cancer and patients’ quality of life.The American cancer society estimates that more than 90 percent of all cancer deaths are related to multidrug resistance(MDR).Although many factors have been proven to contribute to multidrug resistance,the mechanism of multidrug resistance is not fully understood now.As an effective anti-microtubule drug,paclitaxel can inhibit a variety of tumors,especially breast cancer,and has become a common first-line chemotherapy drug.It can kill breast cancer cells by inhibiting the migration and invasion of tumor cells and inducing apoptosis.Whereas drug resistance often happens during the clinical application of paclitaxel,leading to the recurrence and metastasis of breast cancer.Metformin,a drug for diabetes,research finds that it can not only treat T2 D,but also reduce the patients’ risk of cancer.Diabetic cancer patients treated with metformin during neoadjuvant chemotherapy had a higher pathologic complete remission rate than patients untreated with metformin.Epidemiological and animal studies have shown that metformin exerts anti-neoplastic and chemopreventive effects,increasing cancer cells’ sensitivity to chemotherapy drugs.The main mechanism of antitumor effects of metformin is to activate AMPK at the molecular level.Hippo signaling pathway as an important pathway in regulating cell growth,proliferation and apoptosis,it plays a key role in organ development and tissue regeneration.Its downstream signaling molecule,Yes-associated protein(YAP),usually overexpresses in malignant tumor,participating in tumor proliferation,migration and invasion and epithelial-mesenchymal transitions(EMT),influencing the therapeutic effect of anticancer drugs.It is found that metformin can act on the Hippo signaling pathway and phosphorylate YAP by activating AMPK to inhibit the transcription activity of YAP,and then inhibite tumor cells.And there are increasing evidences that the transcription effects of YAP can promote therapeutic resistance,including chemotherapy,molecular targeted therapy,and radiation therapy.The upregulation of YAP increases the resistance of tumor cells to chemotherapy drugs.It suggests that inhibition of YAP activity may be an effective anticancer strategy.We focus on humanbreast cancer cell lines MDA-MB-231 and MCF-7 to explore the effect of metformin on the biological activity of human breast cancer cells,to investigate the influence of metformin on the anticancer effect of paclitaxel and to explore the role of Hippo signaling pathway in inhibiting breast cancer cells of metformin combined with paclitaxel.Methods:MDA-MB-231、MCF-7 cells were divided into four groups:control group(normal cells,NC),metformin group(MF),paclitaxel group(PTX),and combination group(low concentration metformin combined with paclitaxel,Com).(1)CCK-8 and plate clone formation assay measured the proliferation of MDA-MB-231 and MCF-7 cells.(2)Wound healing assay and transwell chamber migration and matrigel invasion assay measured the migration and invasion of MDA-MB-231 and MCF-7 cells.(3)We used flow cytometry to detecte apoptosis of MDA-MB-231 and MCF-7cells.(4)We detected some proteins of MDA-MB-231 and MCF-7 cells by Western blot.Detection indexs include : AMPK,P-AMPK,YAP,P-YAP,matrix metalloproteinases 2,9(MMP-2,MMP-9),E-cadherin,N-cadherin,etc.(5)Immunofluorescence detected the expression of YAP and P-YAP of MDA-MB-231 and MCF-7 cells.Results :(1)The effects of metformin combined with paclitaxel on the proliferation of MDA-MB-231 and MCF-7 breast cancer cells.CCK-8 assay showed that both metformin and paclitaxel inhibited the proliferation of MDA-MB-231 and MCF-7 breast cancer cells,and the inhibition effect was more significant with the increasing of drug concentration.The IC50 of metformin on MDA-MB-231 and MCF-7 cells was 21 m M(48h)and 13 m M(48h)respectively.The IC50 of paclitaxel on MDA-MB-231 and MCF-7 cells was 10μM(48h)and 0.5μM(24h)respectively.Using different concentration of metformin combined with paclitaxel showed that low concentration metformin could increase the sensitivity of breast cancer cells to paclitaxel and enhance the cytotoxic effect of paclitaxel.The plate clone formation assay also showed that metformin and paclitaxelinhibited the formation of MDA-MB-231 and MCF-7 cell clone,and the effect was more obvious on combination group.(2)The effects of metformin combined with paclitaxel on the migration and invasion of MDA-MB-231 and MCF-7 breast cancer cells.Wound healing assay and transwell chamber migration assay showed that 24 h and 48 h scratch width was significantly reduced compared with 0h in control group,while the width in the experimental groups of 24 h and 48 h had no significantly changes compared with that of 0h.The cell migration distance of 24 h and 48 h in experimental groups was lower than that in control group,and migration distance in combination group was less than that in paclitaxel group..In transwell chamber migration experiment,the migration number of MDA-MB-231 and MCF-7 cells in experimental groups significantly decreased compared with the control group,and the number of cells in combination group was lower than paclitaxel group.In transwell invasion experiment,the number of cells penetrating the basement membrane in experimental groups was lower than that in control group,and combination group was significantly reduced compared with paclitaxel group.(3)The effects of metformin combined with paclitaxel on apoptosis of MDA-MB-231 and MCF-7 breast cancer cells.In low cytometry experiment,apoptosis rate of MDA-MB-231 and MCF-7cells in experimental groups was significantly higher than control group,and that was higher in combination group than paclitaxel group.The difference was more significant in MDA-MB-231 cells than MCF-7cells.(4)The effects of metformin combined with paclitaxel on EMT of MDA-MB-231 and MCF-7 breast cancer cells.The detection of EMT related markers by Western Blot showed that in experimental groups,compared with control group,e-cadherin was highly expressed,and the expression was higher in combination group than paclitaxel group;N-cadherin was lowly expressed,and the expression was lower in combination group than paclitaxel group.MMP-2 and MMP-9,which were associated with invasion and metastasis,were both lowly expressed and the combination group was lower thanpaclitaxel group.(5)The detection of proteins related to Hippo signaling pathway by western blot and cell immunofluorescence.Through western blot experient,we found that there was no significant change of AMPK expression in experimental groups compared with control group,while the expression of P-AMPK was significantly increased,and its expression was higher in combination group than paclitaxel group.The expression of YAP was almost the same in metformin group and control group,and it was higher in paclitaxel and combination group than control group,while the expression level of combination group was lower than paclitaxel group.The expression of P-YAP in experimental groups was higher than control group,and combination group was higher than paclitaxel group.In immunofluorescence experiment,we also found that the expression tendency of YAP and P-YAP were same with western blot experiment.Based on metformin anticancer effect and the role of Hippo signaling pathway in cancer progression,the protein expression differences suggested that metformin may increase paclitaxel cytotoxic effect on breast cancer cells through Hippo signaling pathway.Conclusion:(1)metformin can inhibit the proliferation,migration,invasion and EMT of breast cancer cell lines MDA-MB-231 and MCF-7,and promote cell apoptosis.(2)low concentration metformin combined with paclitaxel can increase the sensitivity of MDA-MB-231 and MCF-7 cell lines to paclitaxel and increase the cytotoxic effect of paclitaxel.(3)Hippo signaling pathway involves in the anticancer effect of metformin combined with paclitaxel.Metformin promotes the phosphorylation of YAP and reduces the expression of YAP,thus increases paclitaxel anticancer effect.